Søren Grove

A novel totivirus and piscine reovirus (PRV) in Atlantic salmon (Salmo salar) with cardiomyopathy syndrome (CMS).

BackgroundCardiomyopathy syndrome (CMS) is a severe disease affecting large farmed Atlantic salmon. Mortality often appears without prior clinical signs, typically shortly prior to slaughter. We recently reported the finding and the complete genomic sequence of a novel piscine reovirus (PRV), which is associated with another cardiac disease in Atlantic salmon; heart and skeletal muscle inflammation (HSMI). In the present work we have studied whether PRV or other infectious agents may be involved in the etiology of CMS.ResultsUsing high throughput sequencing on heart samples from natural outbreaks of CMS and from fish experimentally challenged with material from fish diagnosed with CMS a high number of sequence reads identical to the PRV genome were identified. In addition, a sequence contig from a novel totivirus could also be constructed. Using RT-qPCR, levels of PRV in tissue samples were quantified and the totivirus was detected in all samples tested from CMS fish but not in controls. In situ hybridization supported this pattern indicating a possible association between CMS and the novel piscine totivirus.ConclusionsAlthough causality for CMS in Atlantic salmon could not be proven for either of the two viruses, our results are compatible with a hypothesis where, in the experimental challenge studied, PRV behaves as an opportunist whereas the totivirus might be more directly linked with the development of CMS.

Atlantic salmon bath challenged with Moritella viscosa - Pathogen invasion and host response

The Gram-negative bacterium Moritella viscosa is considered to be the main causative agent of winter ulcer, a disease that primarily affects salmonid fish in sea water during cold periods. The disease is initially characterised by localised swelling of the skin followed by development of lesions. To gain more knowledge of the role of M. viscosa in the pathogenesis of winter ulcer, 159 Atlantic salmon (80-110 g) were exposed to a bath challenge dose of 7 x 10(5) cfu ml(-1) for 1 h at 8.9 degrees C. The first mortalities were registered two days post-challenge and the mortality rate increased rapidly. Multi-organ samples were taken throughout the challenge for culture, immunohistochemistry and PCR analysis. Using real-time PCR, M. viscosa DNA was first detected in the gills of all fish examined 2, 6 and 12 h after challenge. From day 2, the bacterium was detected in the muscle/skin, head kidney, spleen and liver. This was in correlation with positive cultured samples and confirmed systemic infection. The early and consistent detection of M. viscosa DNA in gill samples, and less or not in muscle/skin or intestine, could suggest gills as a port of entry for the bacterium. Immunohistochemical analysis using a polyclonal antiserum against M. viscosa demonstrated generalised staining in the lumen of blood vessels and some positive mononuclear cells. The antigens recognised by the antiserum may have originated from extracellular bacterial products and be part of a bacterial invasion strategy. To better understand the immune response in salmon to M. viscosa infection, the expression profiles of the immune genes IL1 beta, C3, ISG15 and CD83 were studied. Increased expression of IL1 beta and C3 was not induced until day 7, which may suggest that M. viscosa might utilize escape mechanisms to evade the hosts immune system by suppressing relevant immune responses.

Sequence Analysis of the Genome of Piscine Orthoreovirus (PRV) Associated with Heart and Skeletal Muscle Inflammation (HSMI) in Atlantic Salmon (Salmo salar)

Piscine orthoreovirus (PRV) is associated with heart- and skeletal muscle inflammation (HSMI) of farmed Atlantic salmon (Salmo salar). We have performed detailed sequence analysis of the PRV genome with focus on putative encoded proteins, compared with prototype strains from mammalian (MRV T3D)- and avian orthoreoviruses (ARV-138), and aquareovirus (GCRV-873). Amino acid identities were low for most gene segments but detailed sequence analysis showed that many protein motifs or key amino acid residues known to be central to protein function are conserved for most PRV proteins. For M-class proteins this included a proline residue in μ2 which, for MRV, has been shown to play a key role in both the formation and structural organization of virus inclusion bodies, and affect interferon-β signaling and induction of myocarditis. Predicted structural similarities in the inner core-forming proteins λ1 and σ2 suggest a conserved core structure. In contrast, low amino acid identities in the predicted PRV surface proteins μ1, σ1 and σ3 suggested differences regarding cellular interactions between the reovirus genera. However, for σ1, amino acid residues central for MRV binding to sialic acids, and cleavage- and myristoylation sites in μ1 required for endosomal membrane penetration during infection are partially or wholly conserved in the homologous PRV proteins. In PRV σ3 the only conserved element found was a zinc finger motif. We provide evidence that the S1 segment encoding σ3 also encodes a 124 aa (p13) protein, which appears to be localized to intracellular Golgi-like structures. The S2 and L2 gene segments are also potentially polycistronic, predicted to encode a 71 aa- (p8) and a 98 aa (p11) protein, respectively. It is concluded that PRV has more properties in common with orthoreoviruses than with aquareoviruses.

Immune responses in Atlantic salmon (Salmo salar) following protective vaccination against Infectious salmon anemia (ISA) and subsequent ISA virus infection

Infectious salmon anemia (ISA) is an orthomyxoviral disease that has had devastating effects on farmed Atlantic salmon. ISA is still a disease resulting in continued loss of revenues and therefore development of effective vaccines is of great importance. Commercial vaccines against ISA are available, but the efficacy is poorly described. There is little information about vaccine-induced immune factors preventing ISA virus (ISAV) infection today. In this study we assessed the protective effects and immunogenicity of vaccines containing three different quantities of the inactivated ISAV antigen. Our findings indicated that immunization induced effective protection in Atlantic salmon with a relative percent survival (RPS) as high as 86. The level of protection was correlated to the amount of ISAV antigen in the vaccine, and fish immunized with high antigen amounts produced detectable ISAV-specific and neutralizing antibodies. While ISAV infection was detectable in non-vaccinated control fish challenged by cohabitation, no infection was detected in fish immunized with high antigen amounts. After challenge, transcriptional analysis of selected immune-related genes demonstrated activation of innate immune responses in ISAV-infected control fish, but not in vaccine protected fish. This study furthers the knowledge about vaccine efficacy and vaccine-induced immunity to ISAV challenge in Atlantic salmon.

Distribution and retention of antigens of Aeromonas salmonicida in Atlantic salmon (Salmo salar L.) vaccinated with a ΔaroA mutant or formalin-inactivated bacteria in oil-adjuvant

In this study we report the differences in distribution and retention of Aeromonas salmonicida antigens after vaccination with two different vaccines. Parr of Atlantic salmon (Salmo salar) were given intraperitoneal injections of either a commercial, monovalent furunculosis vaccine (Apoject) or live, attenuated A. salmonicida (DeltaaroA). Fish were sampled at weeks 2, 4 and 12 post-vaccination and head kidney and spleen were collected. Presence of LPS and 16S rDNA in isolated leukocytes were investigated by immunocytochemistry and polymerase chain reaction (PCR).16S rDNA was detected in head kidney and spleen of all DeltaaroA vaccinated and most Apoject-vaccinated fish at weeks 2 and 4. At week 12, 16S rDNA was detected in none of the DeltaaroA vaccinated fish, but it was detected in head kidney of 75% of Apoject-vaccinated fish. LPS was detected in both vaccination groups at all sampling times, but most frequently in the DeltaaroA vaccinated fish (in head kidney 75-83% vs. 50%, in spleen 58-67% vs. 17-25%).

Vaccine Adjuvants in Fish Vaccines Make a Difference: Comparing Three Adjuvants (Montanide ISA763A Oil, CpG/Poly I:C Combo and VHSV Glycoprotein) Alone or in Combination Formulated with an Inactivated Whole Salmonid Alphavirus Antigen

Most commercial vaccines offered to the aquaculture industry include inactivated antigens (Ag) formulated in oil adjuvants. Safety concerns are related to the use of oil adjuvants in multivalent vaccines for fish, since adverse side effects (e.g., adhesions) can appear. Therefore, there is a request for vaccine formulations for which protection will be maintained or improved, while the risk of side effects is reduced. Here, by using an inactivated salmonid alphavirus (SAV) as the test Ag, the combined use of two Toll-like receptor (TLR) ligand adjuvants, CpG oligonucleotides (ODNs) and poly I:C, as well as a genetic adjuvant consisting of a DNA plasmid vector expressing the viral haemorrhagic septicaemia virus (VHSV) glycoprotein (G) was explored. VHSV-G DNA vaccine was intramuscularly injected in combination with intraperitoneal injection of either SAV Ag alone or combined with the oil adjuvant, Montanide ISA763, or the CpG/polyI:C combo. Adjuvant formulations were evaluated for their ability to boost immune responses and induce protection against SAV in Atlantic salmon, following cohabitation challenge. It was observed that CpG/polyI:C-based formulations generated the highest neutralizing antibody titres (nAbs) before challenge, which endured post challenge. nAb responses for VHSV G-DNA- and oil-adjuvanted formulations were marginal compared to the CpG/poly I:C treatment. Interestingly, heat-inactivated sera showed reduced nAb titres compared to their non-heated counterparts, which suggests a role of complement-mediated neutralization against SAV. Consistently elevated levels of innate antiviral immune genes in the CpG/polyI:C injected groups suggested a role of IFN-mediated responses. Co-delivery of the VHSV-G DNA construct with either CpG/polyI:C or oil-adjuvanted SAV vaccine generated higher CD4 responses in head kidney at 48 h compared to injection of this vector or SAV Ag alone. The results demonstrate that a combination of pattern recognizing receptor (PRR) ligands, such as CpG/polyI:C, increases both adaptive and innate responses and represents a promising adjuvant strategy for enhancing the protection of future viral vaccines.

Infectious salmon anaemia virus (ISAV) in experimentally challenged Atlantic cod (Gadus morhua)

SummaryJuvenile Atlantic cod, Gadus morhua, (6 g) were challenged with infectious salmon anaemia virus (ISAV) either by intraperitoneal (i.p.) injection or by cohabitation with ISA-diseased Atlantic salmon (Salmo salar). Samplings of cod were performed over a period of 45 days and various tissue samples were collected. The presence of ISAV RNA (segment 8) in samples was assessed by both conventional RT-PCR and a competitive quantitative real-time RT-PCR. In the i.p.-challenged group, ISAV RNA was detected in fish from all samplings, i.e. at days 7, 15, 21, 30 and 45 post-challenge. At day 7 post-challenge, all individual fish were positive, and so were the vast majority of individual tissue samples. At later samplings, the fraction of positive brain samples remained high (∼75%). In contrast, the positive fraction of other tissues/organs declined during the experiment. Analysis of positive brain samples by a quantitative real-time RT-PCR analysis showed that the level of ISAV RNA increased significantly (∼20 times) between days 7 and 30 post-challenge and remained high at day 45, indicating that a replication of ISAV had taken place. ISAV RNA was not detected in any control or cohabitation-challenged fish. No abnormal behaviour, clinical disease or, most notably, mortality was observed in any of the challenge or control groups.

Putative virulence genes in Moritella viscosa: activity during in vitro inoculation and in vivo infection.

Moritella viscosa is considered to be the main aetiological agent of winter ulcer disease, primarily affecting farmed salmonid fish in cold marine waters. Transcription profiles of twelve M. viscosa genes, potentially involved in the pathogenesis, were studied during the course of an in vitro cell culture infection assay. Transcription of the same genes was compared in vivo, in head kidney and ulcer tissues of Atlantic salmon challenged with M. viscosa. During the in vitro infection, three putative toxins: a putative repeats in toxin gene (rtxA), a putative cytotoxic necrotizing factor (cnf) and a putative hemolysin increased their transcription significantly with time and coincident with cell rounding. Furthermore, the majority of the genes were stimulated by presence of fish cells and showed higher activity when adhered to fish cells compared to their planktonic counterpart. In vivo gene transcription studies revealed an up-regulation of a putative lateral flagellin in ulcer compared to head kidney tissues in the same individual. A similar trend was seen for cnf and a gene encoding a putative protease, indicating a role for these factors in colonization and tissue damage.

Real-time PCR detection of Moritella viscosa, the likely causal agent of winter-ulcer in Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss

We report the development of a real-time PCR protocol for specific detection of Moritella viscosa. This bacterium is considered to be the main aetiological agent in development of winter-ulcer, a disease severely affecting salmonid aquaculture in Norway. From a newly elaborated draft version of the genome of M. viscosa, the tonB gene sequence was selected as a suitable basis for the development of the real-time PCR assay. The real-time PCR demonstrated the presence of M. viscosa DNA sequences in 88.1% of samples collected from 35 outbreaks of winter-ulcer in Norwegian fish farms. In contrast, standard culturing on blood agar identified M. viscosa in only 39.7% of fish. While the culturing method revealed a similar prevalence (26 to 27%) of M. viscosa in kidney and ulcer samples, substantially more ulcer (81.5%) than kidney (49.7%) samples were shown positive by real-time PCR.

Effects of dietary deoxynivalenol or ochratoxin A on performance and selected health indices in Atlantic salmon (Salmo salar)

Post-smolt Atlantic salmon (Salmo salar) were fed with standard feed added one of five concentrations of either pure deoxynivalenol (DON; 0.5-6 mg/kg) or pure ochratoxin A (OTA; 0.2-2.4 mg/kg), or no added toxins for up to 8 weeks. Performance effects (feed intake, feed efficiency, gain, length and condition factor), various clinical biochemical parameters, packed cell volume and vaccination response against Aeromonas salmonicidae were all inversely correlated with DON dose, whereas relative liver weight increased with DON dose. In fish fed OTA, however, the effects at the doses tested were rather small. We observed no effects of OTA exposure on performance parameters, but some clinical biochemical parameters tended to increase with OTA dose primarily at 3 weeks, and compared with controls OTA exposure caused increased mRNA expression of two immune markers in the spleen. No liver histopathological effects were found from DON or OTA exposure. For DON, we derived a BMDL20 of 0.3 mg/kg feed for reduced total protein in plasma, a BMDL5 of 0.5 mg/kg feed for reduced condition factor, and a NOAEL of 1 mg/kg feed for DON. For OTA, a BMDL or NOAEL could not be derived (>2.4 mg/kg).